First Prior Art
Presently, thanks to the international human genome project and efforts of researchers involved in the project, it is definite that the genome DNA sequence of the human species will be completely clarified in coming several years. However, the genome DNA sequence (base sequence (gene arrangement)) concerned is a genome DMA sequence of a specific person, and not that of an individual. The genome DNA sequence of an individual is slightly different from that of the specific person, where substitution, deletion, insertion, and the like of a base may have occurred in a gene. Normally, such a slight difference is not critical and does not cause any trouble in the life of the individual.
However, it has also been clarified that the difference in genome DNA sequence as described above determines the predisposition of an individual. For example, this difference causes predispositions of individuals such as those who are tolerant to alcohol, those who do not mind the heat, and those who have a low body temperature.
In particular, it is recognized that reaction of the body against a drug differs among individuals, and for this reason, the difference in genome DNA sequence as described above is considered as significantly important information from the standpoint of medical treatment. Therefore, it is strongly desired that the difference in DNA sequence among individuals as described above be detected after the coming determination of the DNA sequences of all the human genes by the human genome project. If genetic information on an individual is made available, it is possible to provide medical treatment optimal for the individual.
To detect the slight difference in genome DNA as described above, a conventional DNA base sequence determination method by use of electrophorasis may be employed. However, this method requires an exceedingly long time and therefore is not practical as a method for detecting genetic information on many subjects.
In addition, it has been discovered that for predispositions prone to genetic diseases and cancers, for example, only a slight difference in base sequence (difference of one base pair, for example) has a critical indication. For example, it has been discovered that sickle cell anemia, which is a lethal genetic disease, is caused by mutation of only one base pair. From this point, also, it is clear that the conventional determination method is not practical.
The basics in detection of the DNA sequence of a gene of an individual are that the DNA sequence of a target gene has been determined and that how the gene of the individual is different from the so-called human gene DNA sequence is sought, as in the instance of sickle cell anemia described above.
As a method capable of detecting the above difference in a short time, a technique using a DNA chip has been proposed, and the effectiveness thereof has been presented.
For example, first, 1000 types of single-stranded DNAs slightly different from the human gene DNA sequence (base sequence) are synthesized in advance, and placed on a substrate. One type of DNA is placed on one section of the substrate, and the position is recorded.
Next, DNA of the subject is taken, and the double helix structure of the DNA is released into single-stranded DNAs. The DNA is then cut into pieces of an appropriate length, and the DNA pieces are fluorescence-labeled.
Subsequently, the fluorescence-labeled DNAs are allowed to hybridize (conjugate) with the single-stranded DNAs placed in advance on the substrate.
After excess DNA and fluorescent dye are washed away, any position/section of the substrate that emits fluorescence is detected. The DNA placed in advance in the section that emits fluorescence is determined to be the DNA sequence that has hybridized with the DNA of the subject. In other words, by detecting the position emitting fluorescence, it is clarified in a short time how the DNA sequence of the subject has mutated from the human gene DNA sequence.
In the technique described above, it is comparatively easy to increase the number of types of single-stranded DNAs placed in advance on the substrate to more than 1000. However, in this case, to attain precise testing, 1000 types or more of single-stranded DNAs must be placed on extremely fine sections of a chip allocated for the respective types of DNAs at high and uniform density so that each section has a uniform amount of DNAs. In particular, in the case that the area of the section allocated for each type of single-stranded DNA becomes finer with increase of the number of types of single-stranded DNAs to be placed, it will become necessary to realize the requirement described above by manipulating a trace amount of single-stranded DNAs.
Second Prior Art
Particulates have a large ratio of the surface area to the volume, and therefore exhibit behaviors generally different from materials that are small in this ratio. For example, particulates of an inorganic material such as titanium oxide and zinc oxide have ultraviolet removal function, antimicrobial function, catalytic function, and the like. Among particulates of an inorganic material, those having a diameter of the order of nanometers (ultra-fine particles) are expected to provide a quantum effect.
Such particulates having the above functions have received attention for their use in the industrial field. In particular, as for ultra-fine particles having a diameter of the order of nanometers, it is urgently required to develop a technique for manufacturing devices using the quantum effect in the industrial scale.
Particulates of protein having a diameter of about 10 to 20 nm have received attention for their use for biosensors and the like. In particular, among a variety of protein particles, there exist particles capable of containing an inorganic material inside. Such protein particles are provided with natures of both the inorganic material and the protein particles.
The particulates described above are normally available in the form of a colloid solution. However, the form of a colloid solution is disadvantageous when the functions of the particulates are to be effectively used. Therefore, search has been made for a technique that permits effective use of the functions of the particulates in the industrial field using the colloid solution as a raw material.
At present, as such a technique permitting effective use in the industrial field, placing the particulates on a substrate is considered most effective. Therefore, desired is establishment of a technique in which an idealistic two-dimensional film made of particulates placed regularly at high density can be easily formed on a substrate.
Various techniques have been proposed so far for placing particulates on a substrate. Some of such techniques handling comparatively large particles have even been commercialized.
For example, Nagayama et al. have disclosed the following method in “Formation of Holoferritin Hexagonal Arrays in Secondary Films Due To Alder-Type Transition”, Lanbgmuir 1996, vol. 12, pp. 1836–1839. That is, as shown in FIG. 18, a substrate 11 is put in a solution 18 containing particulates 15 (polystyrene spheres having a diameter of about 1 to 2 μm) dispersed therein, and then gradually lifted in the position vertical to the liquid level, forming a wet film 19 on both surfaces of the substrate 11. In this way, a film made of polystyrene spheres having a diameter of about 1 to 2 μm is formed on the surfaces of the substrate 11.
However, when it is intended to apply the above method to ultra-fine particles having a diameter of about 10 nm, the substrate 11 must be lifted at a very low rate. It is difficult to keep the lifting rate constant when the rate is low. In addition, the array of ultra-fine particles of the film may possibly lose uniformity due to vibration and the like that may be generated during lifting of the substrate 11. For these reasons, it is difficult to apply the above method to ultra-fine particles. To solve this problem, Nagayama et al. disclose a method for forming a two-dimensional crystal film made of protein (ferritin, diameter: about 12 nm). This method will be described with reference to FIG. 16.
FIG. 16 is a view illustrating the method for forming a two-dimensional crystal film made of ferritin. Referring to FIG. 16, first, a platinum blade 21 is placed in the position vertical to the surface of a substrate 11 that is mounted on a base 20. A liquid 16 containing ferritin dispersed therein is then dropped into a small space between the substrate 11 and the blade 21, so that the liquid 16 is held in and around the space (hatched portion in FIG. 16) due to the surface tension of the liquid 16. Thereafter, while the blade 21 is kept fixed, the base 20 (that is, the substrate 11) is moved in the direction shown by the arrow at a constant speed (2 μm/sec. in this case). This results in the liquid 16 being applied to the substrate 11. The water content of the liquid 16 is evaporated gradually as the liquid 16 is sequentially applied to the substrate 11, allowing formation of a thin film 22 made of ferritin. The thin film 22 has a thickness of about 10 layers of ferritin particles.
Problems to be Solved
If the above requirement described in relation with the first prior art fails to be realized, the DNA chip causes various problems. To state specifically, if the density of DNAs placed in a certain section is too low, the intensity of fluorescence emitted from hybridized DNAs decreases, deteriorating the signal to noise (SN) ratio. In other words, the fluorescence from hybridized DNAs may possibly be buried in background fluorescence inevitably generated.
Moreover, if the absolute amount of DNAs placed varies with the sections, a plurality of sections may emit fluorescence at different intensities when the DNA of the subject hybridizes in two or more sections. In this event, it is unknown why the fluorescence intensity is low in one section compared with that in another section. Specifically, it is difficult to determine whether the fluorescence intensity is low because the absolute amount of DNA placed is small or because the absolute amount DNA placed is so large that emission of fluorescence is allowed despite of weak non-specific adsorption. This may results in mistake of the determination. Furthermore, the variation in the absolute amount of DNAs placed among chips indicates that the reproducibility of the chip quality is poor. This may results in generation of defective DNA chips.
To overcome the above problems, it is necessary to place types of DNAs on the substrate at high density (about 1012 pcs./cm2) by a uniform amount for each type.
As for the second prior art, in order to realize uniform-quality particulate films with high reproducibility by use of the technique disclosed by Nagayama et al., it is necessary to ensure the movement of the base 20 (that is, the substrate 11) while maintaining a constant ultra-low speed of 2 μm/sec. However, in this movement maintaining a constant ultra-low speed, the speed tends to be greatly influenced by a subtle variation in the environment. For example, the moving speed changes with a slight vibration in the environment. A fluctuation in an atmosphere (such as wind and operator's respiration) changes the amount of evaporation of the ferritin solution. By these changes, the reproducibility of the quality of the particulate films deteriorates. It is therefore necessary to provide a means for ensuring the movement of the base 20 while maintaining a constant ultra-low speed and a means for keeping the surrounding atmosphere constant. However, it is not easy to actually provide these means. Therefore, the method disclosed by Nagayama et al. finds difficulty in providing particulate films with a uniform quality, and thus is not suitable for applications to formation of a particulate film over a large-area substrate and to mass production of particulate films.
In addition, using a large-size blade 21 made of platinum costs high. However, if the blade 21 is made of a material other than platinum, it may possibly be corroded. Moreover, it is very difficult to produce a large-size rigid blade having nanometer-order surface precision.
Other methods have also been proposed, including a method in which a substrate surface is treated in various ways and a particulate film prepared in advance is transferred to the substrate surface (Japanese Laid-Open Patent Publication No. 8-155379), a method in which amphiphilic molecules such as casein molecules are used as a binder and a particulate thin film is automatically formed on the binder (Japanese Laid-Open Patent Publication No. 8-229474), and a lithographic method using a particulate film as a substrate (Japanese Laid-Open Patent Publication No. 8-234450). However, all of these methods are not suitable for mass production.